PROJECT SUMMARY/ABSTRACT A fundamental but poorly understood process in eukaryotic cells is how cells structure their genomes into distinct functional domains. This project addresses this gap in knowledge by studying the centromere, a specific chromatin domain found in all eukaryotes. This stably propagated locus guides the assembly of kinetochores to ensure proper segregation of chromosomes during mitosis and meiosis. Mis-regulation of centromeres adversely affects chromosome segregation resulting in aneuploidy, a condition found in more than 90% of all cancers. Aneuploidy contributes to the development of many diseases, such as cancer and Down syndrome. The goal of this project is to understand the molecular mechanisms underlying the specification and inheritance of centromeres. In most eukaryotes, centromeres are epigenetically governed by the centromere-specific histone H3 variant, CENP-A. CENP-A partially replaces canonical histone H3 at centromeres, and provides the foundation for the assembly of kinetochores. Centromeres are usually embedded in epigenetically distinct heterochromatin, the transcriptionally silenced chromatin domain. Assembly of CENP-A at centromeres is cell cycle-regulated. Parental CENP-A is partitioned equally among daughter centromeres following DNA replication, whereas loading of newly synthesized CENP-A to centromeres is uncoupled from DNA replication. How CENP-A chromatin at centromeres is assembled throughout the cell cycle remains poorly understood. Mislocalization of CENP-A to non-centromeric regions has a devastating impact on chromosome segregation, and has been linked to a variety of cancers. Ubiquitin- mediated proteolysis of CENP-A is a conserved mechanism to prevent CENP-A mislocalization. But how non- centromeric regions are protected from CENP-A mis-incorporation in normal cells is largely unexplored. In addition, CENP-A in centromeres is interspersed with the canonical histone H3. The histone H3 within centromeres is actually vital for proper assembly of CENP-A chromatin. How CENP-A and H3 levels are properly balanced in centromeres is unknown. We propose to use fission yeast (Schizosaccharomyces pombe) to address these outstanding questions. Fission yeast is a simple eukaryotic model organism with many aspects of centromere regulation conserved with humans. It is particularly suited to an interdisciplinary approach that includes genetics, genomics, cytology, biochemistry, and structural biology. We propose to: 1) define the mechanisms underlying cell cycle-dependent CENP-A assembly at centromeres, 2) determine how formation of ectopic CENP-A chromatin is prevented, 3) identify regulatory mechanism for how CENP-A and histone H3 levels are balanced at centromeres. Our study also provides important new insights into the role of heterochromatin in centromere function. Given that epigenetic regulation in fission yeast is conserved, our studies will shed light on the processes governing chromosome segregation in human cells, and contribute to a better understanding of human diseases resulting from centromere misregulation.